Staphylococcus aureus causes persistent and recurrent infections, even when highly active antibiotics are used. Dr. Proctor recently reported that an electron transport deficient subpopulation of organisms can be responsible for antibiotic failures and for late recurrences with S. aureus. Biosynthetic defects are present in this subpopulation which make them menadione or hemin auxotrophs. Menadione is the precursor for menaquinone and hemin is used in cytochromes: components in the electron transport chain; making these strains electron transport variants (ETVs). Dr. Proctor has observed that ETVs resist antibiotics and persist within cultured endothelial cells because they produce little alpha-toxin. Uptake of many positively charged antibiotics (aminoglycosides, many lantibiotics, cationic peptides) require intact electron transport to generate an electrochemical gradient. Also, ETVs grow very slowly, due to low levels of ATP which is required for cell wall biosynthesis, thus they are more resistant to cell wall antibiotics. In prospective studies of patients with cystic fibrosis and with gentamicin beads placed for treatment of S. aureus osteomyelitis, ETVs were frequently found. Clinical microbiology laboratories may overlook ETVs because of their slow growth (pinpoint colonies at 24-48 hrs on solid medium and overgrown in liquid medium) and misidentify ETVs because they are atypical (slowly coagulase positive, non-hemolytic, mannitol negative, non-pigmented). Missing this subpopulation has significance because they are more antibiotic resistant than their parent strain, hence, a major reporting error is made (false susceptibility). While all of these phenotypic changes might be explained by defects in biosynthesis of components of the electron transport chain, these observations have been made in genetically undefined strains. Therefore, the PI proposes to construct defined mutations in genes controlling hemin and menadione biosynthesis to test the hypothesis that defects in electron transport allow for intracellular persistence, and cause increased antibiotic resistance. The PI will test mutants for persistence and resistance to antibiotics in cultured cells and in an in vivo soft tissue infection model. The PI will also genetically characterize several clinical ETVs to demonstrate that their phenotype arises from a defect in the biosynthesis of an electron transport chain component. Providing data to support this hypothesis will: i) lay the groundwork for clinical trials for more effective treatment of patients who harbor ETVs, ii) demonstrate the alterations in metabolism can profoundly effect expression of virulence factors, and iii) provide a strong basis for development of compounds that can alter formation of ETVs. For example, with overwhelming sepsis, rapidly shutting off virulence factors is desirable; whereas preventing ETV formation may allow for more effective therapy of chronic infections.